Studies on the cytochrome b 6 / f complex. I. Characterization of the complex subunits in Chlamydomonas reinhardtii

Abstract

We have analyzed the heme-associated peroxidase activity in thylakoid membranes from the. green algae Chlamydomonas reinhardtii after electrophoresis in the presence of sodium dodecyl sulfate. Besides cytochrome f and cytochrome b6, we observed peroxidase activity in two other bands, of 34 and 11 kDa, of unknown origin. Characterization of the b6/f complex subunits was undertaken by means of a comparison of the polypeptide deficiencies in several b6/f mutants with the polypeptide content of preparations enriched in b6/f complexes. We conclude that the b6/f complex consists of five subunits. Using site-specific translation inhibitors, we show that cytochrome f, cytochrome b 6 and subunit IV are of chloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmic ribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunits of chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of the subunits encoded in the nuclear genome.

A New Subunit of Cytochromeb 6 f Complex Undergoes Reversible Phosphorylation upon State Transition

“…Sequence and Topology of the PETO Gene Product-We have previously identified a nucleus-encoded polypeptide of 19-kDa apparent molecular mass that was absent from the thylakoid membranes of cytb 6 f mutants (21). To avoid its copurification with some ATP synthase subunits that display a similar apparent molecular mass, we recovered suV (PetO) from the ATP synthase-deficient mutant F54, using a thylakoid membrane solubilization with Mega-8, which selectively extracts cytb 6 f complex.…”

“…A Transmembrane Protein Associated with the cytb 6 f Complex-We first identified suV as a nucleus-encoded polypeptide of apparent molecular mass of 19 kDa that was present in cytb 6 f-enriched fractions and deficient in thylakoid membranes of C. reinhardtii mutants lacking the cytb 6 f complex (21). We therefore proposed that suV was a genuine subunit of the cytb 6 f complex.…”

“…Last, a nucleus-encoded 19-kDa polypeptide, termed suV, had been proposed to associate with the rest of the cytb 6 f subunits in C. reinhardtii, based on its presence in cytb 6 fenriched fractions and its absence or low representation in cytb 6 f-deficient mutants (21).…”

“…For polypeptide analysis, samples were resuspended in 100 mM 1,4-dithiothreitol and 100 mM Na 2 CO 3 and solubilized in the presence of 2% SDS at 100°C for 50 s. Polypeptides were then separated on 12-18% SDS-polyacrylamide gels in the presence of 8 M urea, cells, and thylakoid membranes were loaded at chlorophyll constant. Heme staining was detected by peroxidase activity of heme-binding subunits using 3,3Ј,5,5Ј-tetramethylbenzidine as described previously (21). Immunodetection using antisera raised against cytf, suIV, Rieske protein in combination with 125 I-labeled protein A, or an enhanced chemiluminescence method was carried out as in Ref.…”

Functional Analysis of a Divergent System II Protein, Ccs1, Involved in c-Type Cytochrome Biogenesis

“…Strains and Culture Conditions-C. reinhardtii wild-type strain CC-125 (MTϩ) and mutant strains ccsA-B6 (CC-2695/CC-2934), ccs1-ac206 (CC-939/CC-1112), ccs1-2 (CC-3422/3423), ccs1-3 (CC-3424/CC-3425), ccs1-4 (CC-3426), abf3 (now ccs1-5::NIT1), ccs2-1 to ccs2-5 (CC-3428 to CC-3437), ccs3-F18 (CC-3092/CC-3093), ccs4-F2D8 (CC-3910/CC-3720), and ccs5-1::ARG7 (CC-3717/CC-3718), described previously (4,43,45,47,56,57), can be obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC). Arginine-auxotrophic strain arg7cw15A used for insertional mutagenesis was obtained from Prof. J.-D. Rochaix, University of Geneva, Switzerland.…”